Registration Dossier

Administrative data

Description of key information

2-(2-ethoxyethoxy)-2-methylpropane was not a skin sensitiser in the LLNA. [BASF, 2013]

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-07-2013 to 2013-10-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test: 9 - 10 weeks (beginning of treatment), Main study: 10 - 11 weeks (beginning of treatment)
- Weight at study initiation: 20.8 +/- 1.2
- Housing: group
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 50-94 %
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6 p.m.

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 % in acetone/olive oil and 100 % (undiluted test item)
No. of animals per dose:
5 (20 animals for the whole test)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429
- Irritation: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals.
- Lymph node proliferation response: At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Thus, the test item in the main study was assayed at 25%, 50% (w/w), and 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node assay
- Criteria used to consider a positive response: The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice.


TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25% and 50% in acetone:olive oil (4+1, v/v), and 100% (undiluted test item). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone. Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.8 μCi of 3H-methyl thymidine (equivalent to 79.0 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test was used for detection of possible outliers (performed with Microsoft Excel 2007). However, both biological and statistical significance were considered together.
Positive control results:
0 % (Test item concentration) : DPM = 2201, SI = 1.0
5 % ((Test item concentration): DPM = 3518, SI = 1.6
10 % (Test item concentration) : DPM = 5251, SI = 2.4
25 % (Test item concentration) : DPM = 12915, SI = 5.9
Parameter:
SI
Remarks on result:
other: Control group: SI = 1.00 25 %: SI = 1.11 50 %: SI = 1.04 100 %: SI = 1.38
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control group: Mean DPM = 241.5 25 %: Mean DPM = 268.9 50 %: Mean DPM = 251.5 100 %: Mean DPM = 333.3
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle acetone:olive oil (4+1, v/v). The local lymph node assay is recommended by international test guidelines as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations. For this purpose a local lymph node assay was performed using test item concentrations of 25%, 50% (w/w), and 100% (undiluted test item). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment). The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in ear weights was also not observed in any treated group in comparison to the vehicle control group. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.11, 1.04 and 1.38 were determined with the test item at concentrations of 25% and 50% (w/w) in acetone:olive oil (4+1, v/v), and 100% (undiluted test item), respectively. A dose response was not observed. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight was not observed in all dose groups in comparison to the vehicle control group. A statistically significant increase in lymph node cell counts was observed in the high dose group (p < 0.003). Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was slightly exceeded in this dose group. However, these findings were not biologically relevant, as all DPM values were neither significantly increased compared to vehicle control group nor did any dose group produce a S.I. of 3 or higher compared to vehicle values. The test item was thus not a skin sensitiser under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

no information available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008:

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation No 605/2014.